Blood pressure is a function of the total volume of blood and the resistance to its flow through the circulatory system, which in turn is determined by the degree of constriction of small blood vessels called arterioles. Hypertension, characterized by an abnormally high arterial blood pressure relative to normal blood pressure (below 140 mm Hg systolic, 90 mm Hg diastolic), is a frequent and dangerous pathological condition that affects a large number of people throughout the world. The prevalence of hypertension may be as high as 60 million adults in the United States alone. Unfortunately, the disease remains undetected in large segments of the population. Low-renin hypertension, which constitutes about one-quarter of the total cases of hypertension in man, is characterized by expanded blood volumes and low concentrations of renin, a protein-digesting enzyme released by the kidney that initiates the production of angiotensin, a potent constrictor of blood vessels.
The prevalence of definite hypertension (systolic blood pressure greater than 160 mm Hg and diastolic pressure greater than 95 mm Hg) is 18.1 per 100 adults in the age group of 18 to 74 years (Roberts and Maurer, "Blood Pressure Levels of Persons 6-74 years, United States, 1971-1974", Vital Health Stat. 203:1 (1977)). Borderline hypertension, defined as blood pressure above 140 mm Hg systolic and 90 mm Hg diastolic, but less than 159 mm systolic and 94 mm diastolic, has been found in approximately 25 million persons (Krishan, "Hypertension: Definition, Prevalence and Prognosis", in Spittell, "Clinical Medicine", Harper and Row, Philadelphia, Pa., (1986)). Hypertension is a leading contributor to cardiovascular disability including stroke, congestive heart failure (CHF) and coronary artery disease, sometimes resulting in premature death. There are a few well-defined causes of hypertension, including kidney disease, narrowing of kidney arteries ("renovascular hypertension") and certain endocrine conditions associated with high circulating hormone levels, e.g. pheochromocytoma (due to excessive epinephrine and/or norepinephrine levels) and aldosteronoma (due to excessive aldosterone levels).
The presence of excess amounts of sodium chloride, i.e. "salt", has long been implicated in the pathogenesis of hypertension (see Dahl, "Salt and hypertension", Am. J. Clin. Nutr. 25:231 (1972)). Increased amounts of sodium are thought to result in excess fluid volume that interferes with normal physiology. In addition, it has been suggested that potassium may modulate the effects of sodium in the development of hypertension. Recent evidence suggests that hormones may be the link between excessive dietary salt intake and at least some forms of hypertension. These hormones, called "natriuretic" (salt-losing) hormones are thought to act on sodium ion transport first on the kidneys, increasing the excretion of sodium ions into the urine, and, secondly, on other cells of the body. Sodium ion transport is performed by a "pump" utilizing the energy released during the hydrolysis of adenosine triphosphate (ATP) by the enzyme sodium, potassium activated adenosine triphosphatase (Na-K-ATPase, E.C. 3.6.1.3). The hypothalamic-renal form of natriuretic hormones are thought to inhibit this enzyme, i.e. act as circulating sodium pump transport inhibitors.
One theory suggests that hypertension results from the effect of the circulating sodium pump transport inhibitors on vascular endothelial transport of sodium and calcium, resulting in retention of calcium within the vascular endothelial cell, and hence enhanced vascular contractility (Blaustein, "Role of a natriuretic factor in essential hypertension: An hypothesis" Ann. Int. Med. 898:785-792 (1983)). A later study showed that low molecular weight inhibitors (&lt;1K daltons) in urine induce or potentiate vasoconstriction in isolated rabbit blood vessels (Weber et al., "Effects of a human-derived sodium transport inhibitor on in vitro vascular reactivity" J. Hypertension 2(10):754-761 (1989). One of the low molecular weight substances was observed to be more potent than the other urine fraction in inducing spontaneous vasoconstriction and in potentiating vasoconstriction caused by norepinephrine or angiotensin. Use of calcium channel blockers or low calcium bath suggested that the inhibitors exert their effect, at least in part, via control of calcium ion movement in vascular smooth muscle cells. Other researchers have shown similar vasoconstrictive effects of endogenous sodium transport inhibitors (Chiba et al., "Vasoconstrictor effects of endogenous digitalis-like factors extracted from urine of hypertensive patients", Heart Vessels 3(3):129-134 (1987); and Mir et al., "Calcium retention and increased vascular reactivity caused by a hypothalamic sodium transport inhibitor", Clin. Sci. 75: 197-202 (1988)). Thus, natriuretic substances may cause smooth muscle cells in arterioles to contract, raising blood pressure.
Studies in man and animals with experimentally expanded blood volumes have also confirmed that many cases of hypertension are associated with an increased circulating plasma level of one or more sodium transport inhibitors. Increased amounts of natriuretic substances have also been detected in acutely volume-expanded subjects. It has been suggested that volume expansion triggers the brain to initiate the production or secretion of natriuretic substances. Two major volume-responsive natriuretic hormone systems are believed to exist. The first system alluded to above involves a hormone most likely synthesized in the posterior hypothalamus ("hypothalamic-renal natriuretic hormone") (Haupert, "Regulation of Na-K-ATPase by the endogenous inhibitor from hypothalamus", Hypert. (Suppl. I) 10:161-166 (1987)), which acts as a transport inhibitor in anuran membrane systems (Buckalew and Nelson, "Natriuretic and sodium transport inhibitory activity in plasma of volume-expanded dogs", Kidney Int. 5:12-22 (1974)), inhibits Na-K-ATPase (Gonick et al., "Circulating inhibitor of sodium-potassium-activated adenosine triphosphatase after expansion of extracellular fluid volume in rats", Clin. Sci. 53:329-334 (1977); Gonick et al., "Regulation of Extracellular Volume: Critical evaluation of natriuretic hormones", In Kruck and Thurau, Eds., Endocrine Regulation of Electrolyte Balance, Springer, Berlin, Heidelberg, New York, Tokyo, pp. 104-120 (1986); and Raghavan and Gonick, "Partial purification and characterization of natriuretic factor from rat kidney", Proc. Soc. Exp. Biol. Med. 164(1):101-104 (1980)) and causes a natriuresis (increased sodium excretion into the urine), without attendant kaliuresis (increase in potassium excretion in the urine) or change in renal hemodynamics when injected into test animals (Gonick and Saldanha, "A natriuretic principle derived from kidney tissue of volume expanded rats", J. Clin. Invest. 56:247-255 (1975)).
The second natriuretic hormone, referred to as "atrial natriuretic factor" ("ANF") or "atrial natriuretic peptide" ("ANP") is either synthesized or stored in cardiac atria (deBold "Heart atria granularity effects of changes in water-electrolyte balance", Proc. Soc. Exp. Med. Biol. 161:508-511 (1979)) and does not inhibit active transport (Atlas et al., "Purification, sequencing and synthesis of natriuretic vasoactive rat atrial peptide", Nature 309:717-722 (1984)). At high blood levels, ANF exerts its predominant natriuretic effect via increasing renal glomerular filtration rate (Cogan, "Atrial natriuretic factor can increase renal solute excretion primarily by raising glomerular filtration", Am. J. Physiol. 250:F710-714 (1986)), while at lower blood levels, natriuresis occurs via a direct effect on tubular transport of sodium, without alteration in glomerular filtration rate (Soejima et al., "Renal response of anesthetized rats to low-dose infusion of atrial natriuretic peptide", Am. J. Physiol. 255:R449-R455 (1988)). ANF has been structurally identified (Flynn et al., "The amino acid sequence of an atrial peptide with potent diuretic and natriuretic properties", Biochem. Biophys. Res. Commun. 117:859-865 (1983)) and a radioimmunoassay has been developed (Burgisser et al., "Human cardiac plasma concentrations of atrial natriuretic peptide quantified by radioreceptor assay", Biochem. Biophys. Res. Comm. 133:1201-1209 (1985)). In congestive heart failure, ANF levels have been shown to be increased above normal, reflecting release secondary to dilatation of the cardiac atria (Raine et al., "Atrial natriuretic peptide and atrial pressure in patients with congestive heart failure", New Eng. J. Med. 315:534-537 (1986)). This increase in ANF is physiologically ineffective or only partially effective, as demonstrated by the persistence of the avid renal sodium reabsorption characteristic of congestive heart failure.
In contrast to the more recently discovered ANF, the hypothalamic-renal natriuretic hormone (HRNH) has not been purified sufficiently to provide structural information or to permit the development of a radioimmunoassay. Thus, studies of changes in the circulating level of HRNH following physiological manipulations or in human disease states have relied on the use of certain functional properties tested in biological assays; on size (i.e. molecular weight) and conditions under which the hormone is detected to identify and quantify the changes. Natriuretic activity is typically assayed by injecting the putative natriuretic substance into a test animal such as a rat and determining whether sodium excretion into the urine increases in response to the administered substance. In addition to the production of natriuresis in a bioassay animal, the transport inhibitor has been identified by a variety of techniques including: 1) inhibition of short circuit current in anuran membranes ("antinatriferic" activity) (Gruber and Buckalew, "Further characterization and evidence for a precursor in the formation of plasma antinatriferic factor", Proc. Soc. Exp. Biol. Med. 159:463-467 (1978)); 2) direct inhibition of purified Na-K-ATPase ATPase (Hamlyn et al., "A circulating inhibitor of Na.sup.+ associated with essential hypertension", Nature 300:650-652 (1982)); 3) displacement of tritiated ouabain from receptors on purified Na-K-ATPase (Kelly et al., "Characterization of digitalis-like factors in human plasma. Interactions with Na-K-ATPase and cross-reactivity with cardiac glycoside-specific antibodies", J. Biol. Chem. 260:11396-11405 (1985)) or red cell membrane Na-K-ATPase (Devynck et al., "Measurement of digitalis-like compound in plasma: application in studies of essential hypertension" Br. Med. J. 287:631-634 (1983)); 4) inhibition of active transport in isolated membranes (Buckalew and Nelson, supra), or cells (Edmonson et al.; Haddy et al., De Wardener and MacGregor; Blaustein and Kelly et al., supra); and 5) cross-reactivity with digoxin antibodies (Kelly et al., supra). This last technique may be the least discriminating because significant discrepancies have been observed in serum or urine fractions which show enhanced Na-K-ATPase inhibitory activity or natriuretic activity but not digoxin immunoreactivity, or the reverse (Kelly et al., supra; Weiler et al., "Observations on the "cascade" of Na-K-ATPase inhibitory and digoxin-like immunoreactive material in human urine: Possible relevance to essential hypertension", Clin. Exp. Hypertension [A]7 (5 and 6):809-836 (1985); Kramer et al., "Further characterization of the endogenous natriuretic and digoxin-like immunoreactivity activities in human urine:effect of changes in sodium intake", Renal Physiol. 8:80-89 (1985) and Crabos et al., "Measurement of endogenous Na.sup.+, K.sup.+ -ATPase inhibitors in human plasma and urine using high-performance liquid chromatography", FEBS Letters 176:223-228 (1984); Klingmuller et al., "Digoxin-like natriuretic activity in the urine of salt loaded healthy subjects", Klin. Wochenschr. 60(19)1249-1253 (1982)).
In at least one study which reported an increased level of Na-K-ATPase inhibitory activity in urine fractions of hypertensives as compared to normals, no parallel changes were found in digoxin immunoreactivity (Weiler et al., supra). On the other hand, several non-humoral agents in plasma have been found to be potent Na-K-ATPase inhibitors, including non-esterified fatty acids and lysophosphatidyl choline (Kelly et al., "Identification of Na, K-ATPase inhibitors in human plasma as nonesterified fatty acids and lysophospholipids", J. Biol. Chem. 261:11704-11711 (1986)). Thus, great care must be taken to evaluate plasma inhibitors of Na-K-ATPase to exclude all non-specific inhibitors.
Attempts to purify NH materials have resulted in putative NH compounds having one or more of the above-enumerated activities and of varying molecular weights. When Na-K-ATPase inhibitory activity is used to bioassay NH, several low molecular weight forms have been detected in deproteinized plasma following separation by HPLC. One study reports three low molecular weight fractions in desalted, deproteinized human plasma, separated by HPLC, which displayed Na-K-ATPase inhibitory activity, inhibited sodium pump activity in erythrocytes, displaced .sup.3 H-ouabain from canine kidney Na-K-ATPase, and cross-reacted with polyclonal and monoclonal digoxin-specific antibodies (Kelly et al., 1985, supra). A fourth fraction cross-reacted with digoxin antibodies, but was not an Na-K-ATPase inhibitor.
Crabos et al., supra, also sought endogenous Na-K-ATPase inhibitors in HPLC-separated, deproteinized plasma and urine from hypertensive and normotensive urines. Three Na-K-ATPase inhibitors were found in plasma and two inhibitors in urine. Of the plasma fractions, two showed equal potency in a digoxin radioimmunoassay, while one showed minimal cross-reactivity with digoxin antibody. Two of the inhibitors were also present to a greater degree in plasma of hypertensive patients than in plasma from normotensive controls. A fourth plasma fraction reacted with digoxin antibodies, but did not inhibit Na-K-ATPase. Cloix et al., ("High-yield purification of a urinary Na pump inhibitor", Biochem. Biophys. Res. Commun. 131:1234-1240 (1985)) have reported purification to homogeneity of a low molecular weight Na-K-ATPase inhibitor from human urine. This compound appears to be a glycosteroid with molecular weight less than 500 daltons. Gruber et al. described two low molecular weight Na-K-ATPase inhibitors in plasma of volume expanded dogs that also cross-react with digoxin antibody (Gruber et al., "Further characterization and evidence for a precursor in the formation of plasma antinatriferic factor", Proc. Soc. Exp. Biol. Med. 159:463-467 (1978) and Gruber et al., "Endogenous, digitalis-like substance in plasma of volume-expanded dogs", Nature 287:743-745 (1980)).
In general, these putative NH materials have molecular weights of less than 1000 and some show cross-reactivity with digoxin antibodies. Less attention has been focused on high molecular weight compounds (10,000 to 50,000 daltons) from plasma or urine, which have the properties of inducing natriuresis or inhibiting the Na-K-ATPase pump. These compounds have not been well characterized physiologically or biochemically.
Sealey et al., ("Natriuretic activity in plasma and urine of salt-loaded man and sheep", J. Clin. Invest. 48:221-224 (1969)) first described the presence of a high molecular weight natriuretic compound in urine and deproteinized plasma from salt-loaded man and sheep. Clarkson et al. ("Two natriuretic substances in extracts of urine from normal man when salt-depleted and salt-loaded", Kidney Int. 10:381-394 (1976)) reported that both high and low molecular weight natriuretic compounds could be recovered from urine of salt-loaded man. The high molecular weight compound characteristically had a delayed onset of action and produced a relatively long duration of natriuresis, while the low molecular weight compound had an immediate onset and shorter duration of action.
High molecular weight proteins have also been demonstrated in plasma from hypertensive animals and humans. Researchers have described a 14 to 15 kD protein band in plasma of Dahl salt-sensitive rats, which increased in intensity as hypertension developed with salt loading, but was much less affected by salt loading in Dahl salt-resistant rats (Morich and Garthoff ("Characteristic changes of plasma proteins in the Dahl hypertensive rat strain (DS) during the development of hypertension", Hypertension 3:1249-253 (1985)). Additional work characterized this "hypertension associated protein" as having a molecular weight of approximately 15,500 daltons and as the alpha 1-chain of haptoglobin (John et al., "Identification of a so-far not characterized human serum protein associated with essential hypertension", Electrophoresis 6:292-295 (1985)). Cloix et al., ("Plasma protein changes in primary hypertension in humans and rats" Hypertension 5:128-134 (1983)), found a similarly sized protein (molecular weight 13,000 daltons) in plasma from hypertensives (87%) as contrasted to 26% of normotensives without history of hypertension. Van de Voorde et al., "Isolation of a plasma protein observed in patients with essential hypertension", Biochem. Biophys. Res. 111:1015 (1983), found a 105 kD protein in plasma of essential hypertensives that could be broken down to two 45 kD and one 15 kD components.
In recent studies, Weiler et al., "Circulating High Molecular Weight Form of Na-K-ATPase Inhibitor: Changes in Disease", Clin. Res. 34:90A (1986) isolated a protein band of molecular weight of approximately 10,000 daltons in plasma and compared the presence of the band in plasma from patients with primary aldosteronism, chronic renal failure, congestive heart failure and normals. The results demonstrated increased intensity of staining of the band in primary aldosteronism and chronic renal failure, and decreased intensity of the band in congestive heart failure, indicating that the amount of the protein varied in proportion to "effective circulating blood volume" in disease states.
The majority of the prior investigations of the circulating transport inhibitor(s) in hypertension have employed whole plasma or plasma extracts as the source of the inhibitor(s). However, there is evidence that the circulating substance, measured either as a natriuretic compound, or as a Na-K-ATPase inhibitor, exists in several forms both in plasma and urine, including a carrier-bound moiety (Veress et al., "Characterization of the natriuretic activity in the plasma of hypervolaemic rats", Clin. Sci. 59:183-189 (1980); Pearce and Veress, "Concentration and bioassay of a natriuretic factor in plasma of volume expanded rats", Can. J. Physiol. Pharmacol. 53:742-747 (1975); and Weiler et al., "Observations on the "Cascade" of Na-K-ATPase Inhibitory and Digoxin-Like Immunoreactive Material in Human Urine: Possible Relevance to Essential Hypertension", Clin. and Exper Theory and Practice A7(5 & 6):809-836 (1985)), and one or more precursors (Gonick et al., Endocrine Regulation of Electrolyte Balance, supra; and Gruber et al., "Evidence that natriuretic hormone is a cascading peptide hormone system", In Lichardus et al., Eds., Hormonal Regulation of Sodium Excretion, Elsevier/North-Holland, Amsterdam, pp. 349-355 (1980)).
Veress and co-workers ("Characterization of the natriuretic activity in the plasma of hypervolaemic rats", Clin. Sci. 59:183-189 (1980)) have demonstrated that when plasma is not deproteinized and is separated on Sephadex with an eluate of low ionic strength, the natriuretic activity is confined to a large protein fraction (molecular weight &gt;30K daltons), whereas separation with an eluate of high ionic strength yields both high and low molecular weight natriuretic factors. These researchers concluded that natriuretic factor may occur in plasma in both protein-bound and free forms and that deproteinization of plasma or use of a high ionic strength buffer might separate the low molecular weight form from its carrier protein.
In additional studies by Gruber et al., these researchers suggested that the quantity of the final low molecular weight NH present in plasma was a function of the method of processing the plasma sample. Rapid processing of chilled blood collected in the presence of an enzyme inhibitor, bacitracin, with subsequent acidification and boiling, yielded predominantly a "precursor" low molecular weight NH, while plasma processed slowly, without bacitracin, and incubated at room temperature for 30 minutes yielded a "final" low molecular weight NH. These observations provided a hypothesis that a precursor, low molecular weight NH was metabolized to a final low molecular weight NH by proteolytic enzymes and was the major circulating form of the low molecular weight NH.
Thus, in contrast to the majority of previous studies in which plasma is deproteinized by heating and/or acidification prior to assay, these studies suggest that initial deproteinization must be avoided if the presence of high molecular weight natriuretic substances or pump inhibitors are to be sought.
In a study comparing both plasma and urine in normotensives and hypertensives (Gonick et al., "Pattern of Na-K-ATPase inhibitors in plasma and urine of hypertensive patients: A preliminary report", Klin. Wochenschr. 65 (Suppl. VIII):139-145 (1987)), sequential Amicon.RTM. filtration was performed, followed by Sep-Pak.degree. HPLC separation of the low molecular weight (less than 1 kD) fractions. These studies confirmed that hypertensive urine contained increased quantities of a high molecular weight (30 kD to 50 kD) Na-K-ATPase inhibitor and one low molecular weight inhibitor. In plasma (collected in chilled tubes containing proteolytic enzyme inhibitors to avoid denaturation), two low molecular weight plasma inhibitors were increased, but relatively little Na-K-ATPase inhibitory activity was found in the large molecular weight compounds (&gt;1 Kd) in plasma. The low inhibitory activity in the larger molecular weight fractions of plasma suggested that the low molecular weight inhibitor might be masked by association with a carrier protein or incorporation in a precursor molecule that requires further in vivo metabolism before its activity could be expressed. Pre-treatment of the &gt;50 kD plasma fraction with mercaptoethanol followed by application of the plasma fraction to SDS-PAGE, yielded a 12,000 dalton protein band, identical in locus to the Na-K-ATPase inhibitor isolated from mercaptoethanol-treated plasma as described by Weiler et al., Clin. Res., supra.
These reports attest to the difficulty of isolating natriuretic substances from plasma and urine. Because of the variety of procedures used to attempt to purify such substances, verification that a putative natriuretic substance having a particular molecular weight is the "native" compound having biological activity in vivo has not been possible. Correlation of low molecular weight natriuretic substances with disease states such as hypertension, is also hampered by the absence of a protocol that preserves the relationship of low molecular weight substances with "carrier" proteins or precursor proteins that exist in vivo. In addition, comparison of substances isolated from plasma by various researchers, and between substances isolated from plasma with those from urine is tenuous at best. Substances found in the urine may be altered, for example by the kidneys, such that the active form of a NH circulating in the plasma is different from the NH isolated from urine.
Correlation of putative NH substances with various diseases, including hypertension, has been attempted. In essential hypertension, there is preliminary data which point to a relationship between circulating levels of pump inhibitors and body volume status. Hamlyn et al., supra, reported an inverse correlation between the total level of plasma Na-K-ATPase inhibitors and renin activity, which is assumed to be suppressed in the presence of volume expansion. Devynck et al., ("Clinical and biochemical approach of a circulating Na-pump inhibitor", J. Physiol. 79:538-541 (1984)) reported that not only is there an increase in the level of circulating pump inhibitors in the plasma of untreated hypertensives as compared to normotensives, but this increased level is reduced toward normal by treatment with diuretics.
Additional studies suggest an association of the hypothalamic-renal NH with hypertension. For example, plasma from patients with low renin hypertension has been shown to contain more of the pump inhibitor, as measured directly by Na-K-ATPase inhibition (Hamlyn et al., "A circulating inhibitor of Na.sup.+ associated with essential hypertension", Nature 300:650-652 (1982); Huot et al., "Sodium-potassium pump activity in reduced renal mass hypertension", Hypertension 5:94-100 (1983) and MacGregor et al., "Evidence for a raised concentration of a circulating transport inhibitor in essential hypertension", Br. Med. J. 283:1355-1365 (1981)), or indirectly by reduction of ouabain-sensitive sodium efflux from leukocytes (Edmondson and MacGregor, "Leucocyte cation transport in essential hypertension: its relationship to the renin-angiotensin system", Br. Med. J. 282:1267-1269 (1981)), than plasma from other hypertensives and normotensives. Moreover, in animals with various models of experimental hypertension, pump activity in blood vessels (as measured by ouabain-sensitive Rb.sup.+ uptake) is reduced below that of normal controls (Haddy et al., "Humoral factors and the sodium-potassium pump in volume-expanded hypertension", Life Sci. 24:2105-2118 (1979)). Based on such observations, researchers have proposed that essential hypertension may be due to a hereditary defect in the kidney's ability to excrete sodium, thus leading to volume expansion and increased release of natriuretic hormone, which causes vasoconstriction via inhibition of the sodium pump in vascular smooth muscle cells, with an attendant increase in intracellular sodium and calcium. (Blaustein, "Sodium ions, calcium ions, blood pressure regulation and hypertension: a reassessment and a hypothesis", Am. J. Phsiol. 232:C165-C173 (1977); and "Role of a natriuretic factor in essential hypertension: An hypothesis", Ann. Int. Med. 98:785-792 (1983); De Wardener and MacGregor, "The relation of a circulating transport inhibitor (the natriuretic hormone?) to hypertension", Medicine 62:310-326 (1983)).
A few studies have also examined the effects of renal failure and dialysis on the circulating levels of natriuretic compounds or pump inhibitors. Bourgoignie et al. ("The presence of a natriuretic factor in urine of patients with chronic uremia. The absence of the factor in nephrotic uremic patients", J. Clin. Invest. 53:1559-1567 (1974)) reported that gel filtration fractions of serum or urine from patients with chronic renal failure produced a striking natriuresis in test animals. When serum fractions from the same patients in a nephrotic state were tested, no natriuresis was observed. This suggests that the volume status rather than uremia led to accumulation of a natriuretic substance. Boero et al. ("Erythrocyte Na, K pump activity and arterial hypertension in uremic dialyzed patients", Kidney Int. 34:691-695 (1988)) reported that RBC Na, K pump activity was lower in uremic patients than in normals, while serum from uremic patients inhibited ouabain-sensitive sodium efflux in normal RBC. Kelly et al., "Endogenous digitalis-like factors in hypertension and chronic renal insufficiency", Kidney Int. 30:723-729 (1986)) found that levels of digoxin-like immunoreactivity and Na-K-ATPase inhibitory activity in deproteinized plasma were increased in hypertensive patients with mild renal failure, whereas plasma levels in dialysis patients were not different from controls. An observed increase in Na-K-ATPase inhibitory activity was attributed to a rise in two out of three plasma fractions previously described. Devynck et al. ("Circulating inhibitor of sodium active transport in essential hypertension and volemic expansion", Arch. Mal Coeur 78:1691-1695 (1985)), on the other hand, found that plasma levels of a digitalis-like compound (measured by .sup.3 H-ouabain displacement) were elevated in renal failure patients requiring dialysis but these levels were reduced by the dialysis procedure, and the reduction in activity of this compound was proportional to the weight lost during dialysis. Krzesinski et al. ("Arguments for the presence of a Na-K-ATPase inhibitor in the plasma of uremic and essential hypertensive patients", Clin. Exp. Physiol. 79:538-541 (1984)) also reported the presence of a natriuretic factor in plasma of uremic subjects, the activity of which was reduced during dialysis in proportion to weight loss, attesting to the relationship between the circulating levels of this factor and body volume status.
Research conducted by Weiler et al., (Clin. and Exper. Theory and Practice, 1985, supra) compared the pattern of urinary Na-K-ATPase inhibitors in hypertensives and normal controls following sequential Sephadex column and C18 reverse phase separations. The results demonstrated that one, low molecular weight Na-K-ATPase inhibitor was present to an equal degree inhypertensives and normal subjects, whereas an earlier eluting low molecular weight inhibitor and all identifiable larger-sized components were increased in hypertensives. These data suggested that partial inhibition of enzymatic conversion of a precursor low molecular weight NH to the final form might be a factor in some forms of essential hypertension (defined as persistent hypertension without a currently definable etiology). Subsequent work identified a high molecular weight Na-K-ATPase inhibitor in mercaptoethanol-extracted plasma that altered in quantity with disease states (Weiler, Clin. Res., supra). The intensity of a protein band of approximately 10,000 daltons was observed to increase in plasma from patients with primary aldosteronism and chronic renal failure, while decreasing in congestive heart failure. The band intensity returned toward normal after treatment of chronic heart failure. Upon further characterization by extraction and semi-purification on HPLC, one peak which contained the protein band was shown to be a potent inhibitor of Na-K-ATPase. These results demonstrated appropriate directional changes in the quantity of the high molecular weight form of circulating Na-K-ATPase inhibitor in disease states characterized by increased (primary aldosteronism and chronic renal failure) or decreased (congestive heart failure) central volume. In contrast, studies of alpha-h ANP have shown that this natriuretic material increases rather than decreases in CHF (Tikkanen et al., "Plasma atrial natriuretic peptide in cardiac disease and during infusion in healthy volunteers", Lancet 2:66 (1985)), implying that circulating Na-K-ATPase inhibitors may be of greater pathophysiological significance in this disease state.
In further studies, correlation of the high molecular weight inhibitor (approximately 12,000 K daltons) with hypertension has been found (Weiler et al., "Circulating high molecular weight form of Na-K-ATPase inhibitor:changes in disease", Clin. Res. 34:90A (1986); Gonick et al., "High and Low Molecular Weight Plasma Na-K-ATPase Inhibitors (NKAI) in Hypertension, presented at post-congress Satellite Symposium on Natriuretic Hormones in Hypertension, following the Xth International Congress of Nephrology, London, England, August (1987); and Weiler et al., "High Molecular Weight (HMW) Plasma Na-K-ATPase Inhibitor (NKAI) in Essential Hypertension (EH)", abstract, Proceedings of the Third Annual Meeting of The American Society of Hypertension in New York, June 1988 (Amer. J. Hypertension 1(3) Part 2:47A (1988)). In these studies, plasma from patients with essential hypertension, normotensive controls and normotensive controls with a family history of hypertension was first separated by Amicon.RTM. filtration into high and low molecular weight plasma fractions (less than 1,000 daltons). The low molecular weight inhibitors were further separated by C18 Sep-Pak cartridges using a 10% step-wise acetonitrile gradient, while the high molecular weight inhibitors were separated on Sephadex G-75 columns. All fractions were tested for Na-K-ATPase inhibitory activity after pretreatment with formic acid and mercaptoethanol. The concentration of the high molecular weight inhibitor was 30 fold the concentration of the low molecular weight inhibitor, confirming the predominance of the high molecular weight natriuretic material in hypertensive patients. The active fraction of the high molecular weight inhibitors was shown to contain a 12 kD protein band on SDS-PAGE, which was a potent inhibitor of Na-K-ATPase, displaced .sup.3 H-ouabain from its Na-K-ATPase receptor, but did not cross-react with ANF antibody. The high molecular weight inhibitor, but not the low molecular weight inhibitor, correlated positively with diastolic blood pressure and inversely with the natural log of renin. SDS PAGE separation of plasma from these subjects confirmed that patients with essential hypertension had higher staining intensities of the 12 kD compound than normal controls. Normotensive controls with a family history of hypertension had intermediate staining intensities of the 12 kD band. Significant differences were found between the mean concentrations of the high molecular weight inhibitor (12 kD compound) and low molecular weight inhibitors (less than 1 kD) between hypertensive and normal patients. Of the patients with essential hypertension approximately 60% had elevated plasma levels of the 12 kD compound relative to controls. These data clearly demonstrate the key role of the high molecular weight natriuretic hormone in the pathogenesis of hypertension.
With respect to other diseases such as congestive heart failure information is scarce regarding the role of the sodium transport inhibiting natriuretic hormone. Studies have demonstrated that a natriuretic substance found in an ultrafiltrate of normal urine was absent in the urine of patients with congestive heart failure (Kruck and Kramer, "Third factor and edema formation", Contr. Nephrol. 13:12-20 (1978)) and Kramer and Kruck, "Plasma natriuretic activity in oedematous states", Proc. Eur. Dial. Transplant. Ass. 12:321-329 (1976)). These studies also demonstrated that plasma and urine fractions from normals obtained following separation on Sephadex G-25 columns, consistently reduced short-circuit current when applied to the serosal surface of frog skin, whereas plasma and urine fractions obtained from patients with congestive heart failure lacked this effect. These results indicate that patients with congestive heart failure have a deficiency of a natriuretic, transport inhibiting substance which may be the hypothalamic-renal natriuretic hormone.
In view of the apparent association between the presence of natriuretic substances and disease and the long standing difficulty of identification and purification of such substances, it would be useful to provide reproducible methods for purifying Na-K-ATPase inhibiting substances having natriuretic activity that permit correlation with disease states and to provide the substances for diagnostic and therapeutic applications.